Spermatogenesis is an androgen-dependent process requiring intimate contact between the cells that directly respond to the androgenic signal (Sertoli cells) and the cells that ultimately become sperm (germ cells). While the androgen dependence of spermatogenesis has been known for several decades, its molecular basis has remained elusive. The subject of this proposal is an androgen/androgen receptor (AR)-induced transcription factor, RHOX5, which is expressed in Sertoli cells and therefore is a candidate to mediate androgen- dependent events during spermatogenesis. Loss of Rhox5 in mice loss causes increased male germ-cell apoptosis, reduced sperm count, and reduced sperm motility. Because Rhox5 is expressed in Sertoli cells, this suggests that Rhox5 regulates the transcription of genes encoding cell surface and secreted molecules in Sertoli cells that in turn regulate the survival and maturation of the adjacent germ cells. Microarray analysis has identified many genes regulated by Rhox5, including Unc5c, which encodes a pro-apoptotic cell-surface protein, and insulin-2, which encodes a secreted molecule that promotes metabolism and cell survival. Insulin-2 is a direct target of RHOX5 whose expression during the first wave of spermatogenesis depends on Rhox5. Other Rhox5-regulated genes include those encoding the key metabolic regulators PPAR?, PGC1?, resistin, and adiponectin, as well other members of the X-linked Rhox homeobox gene cluster. One of the Aims of this proposal is to begin to define the transcriptional network under the control of RHOX5, in part by using new technology to rapidly screen for more direct targets of RHOX5. These targets, including secondary androgen-response genes, will be characterized and their interrelationships with genes indirectly regulated by Rhox5 will be analyzed in order to begin to establish a Sertoli-cell transcriptional network. The Rhox5 gene is also a model system for elucidating molecular mechanisms that control gene transcription. The other Aim of this proposal focuses on a novel regulatory mechanism that controls the expression of Rhox5's proximal promoter (Pp), a promoter that recruits polymerase II (Pol II) and begins transcriptional elongation in all tissues but only completes transcriptional elongation to allow the production of RHOX5 protein in Sertoli and caput epididymal cells. Preliminary data indicates that completion of Pp transcriptional elongation depends, in part, on AR. This suggests a new function for this nuclear hormone receptor, as AR is classically thought to regulate gene expression only by recruiting Pol II and initiating transcription. In this application, we propose to elucidate the underlying molecular mechanism for this novel mode of regulating gene expression. PUBLIC HEALTH RELEVANCE: The reproductive homeobox-5 (Rhox5) gene encodes a transcription factor. Because it is testosterone regulated and required for the normal fertility of male mice, studying Rhox5 has the potential to illuminate transcription networks responsible for hormone-regulated gene expression and fertility. In addition, Rhox5 is regulated by a novel transcriptional mechanism that may significantly add to our understanding of gene regulation in general.